showed decrease in expression during differentiation indicating neuronal maturation (Fig 2H). Specifically, neural stem cell markers HES5 and DACH1 showed a significant reduction from day 8 to day 49 indicating loss of neural stem cell identity. Finally, we performed immunocytochemical analysis at day 49 for markers of both lineages that showed presence of deep layer CTIP2-, upper layer SATB2-, CGE lineage PROX1-, LGE lineage MEIS2- and interneuron subpopulation marker Calbindin-positive neurons (Fig 2I–2M). To confirm that, all the neurons present in the cultures were human-derived we did a co-stain for human nuclear (HN) marker with MAP2 and astrocytic marker GFAP (Fig 2N). Taken together, RNA profiling of the developing neuronal cultures and immunocytochemistry at end stages indicate the emergence of interneurons of GE lineages, as well as glutamatergic neurons reminiscent of different neocortical layers.
